Sex differences in the incidence and severity of influenza A virus infection have been documented in humans. Although exposure rates are often higher in men, fatality following exposure to pathogenic influenza A viruses is reportedly higher in women. Sex differences also are reported in response to influenza virus vaccines, with women consistently mounting higher antibody responses and developing more frequent and severe side effects following vaccination than men. Small animal models are critical for establishing the mechanisms mediating why males and females respond differently to influenza virus infection and vaccination. Following primary inoculation with the mouse-adapted influenza A viruses A/PR/8/34 (PR8;H1N1) or A/HK/68 (HK68;H3N2), female mice mount higher inflammatory and humoral immune responses than males. Our preliminary data further reveal that elevated immunity in females against influenza A viruses represents a delicate balance between immune responses conferring protection or causing pathology. The goal of this proposal is to develop a small animal model to test the hypothesis that protective immunity to heterosubtypic influenza A virus challenge differs between the sexes and is modulated by sex steroid hormones. In Specific Aim 1, we will establish whether neutralizing antibody responses, virus-specific T cell responses, and protection against lethal influenza A virus challenge is greater among females than males. Whether males and females differentially rely on subsets of adaptive immune cells for protection against lethal influenza A virus infection has not been documented;thus, we also propose to compare heterosubtypic immune responses between male and female mice devoid of specific adaptive immune cell populations. If protective heterosubtypic immunity is elevated in females compared with males, then estrogens and/or progestins may enhance and androgens may suppress adaptive immunity against heterosubtypic influenza A virus challenge. In Specific Aim 2 we will test this hypothesis by manipulating sex steroid concentrations in vivo and establishing the effects on humoral and cell-mediated immunity as well as protection from lethal influenza A virus challenge. These are a series of high risk-high return experiments because there are no data to date assessing the sex-specific induction of heterosubtypic immunity in response to influenza A virus infection. Demonstrating that females mount a broadly protective immune response, however, will have important implications for dealing with annual epidemics of influenza, as this may explain why the attack rates for influenza are higher in men than in woman and influenced by pregnancy. PUBLIC HEALTH RELEVANCE: Sex differences in the incidence and severity of influenza A virus infection as well as in response to vaccination have been documented in humans. Small animal models are critical for establishing the mechanisms mediating why males and females respond differently to influenza virus infection and vaccination. We will evaluate whether higher humoral immune responses following sublethal infection confers greater protection from challenge with pathogenic influenza A viruses in females compared with males and the extent to which these differences are mediated sex steroids, which may provide clues into why responses to pandemic influenza A viruses differ between the sexes and during pregnancy.